Abstract The overall goal of this proposal is to understand and target cancer cell addictions to stress- tolerance pathways. We have discovered that cancer cells maintain proliferation by engaging in a pathway known as translesion synthesis (TLS). By employing TLS, cancer cells are able to replicate in a continuous manner and subvert the replication stress response. Here, we propose to implement state-of-the-art assays to analyze how cancer cells alter DNA replication fork dynamics and replisome components to promote TLS. By defining the core TLS machinery, we will seek to identify biomarkers and novel targets of TLS. Importantly, we have developed a small molecule inhibitor of TLS that selectively halts DNA replication in several cancer cell lines that are dependent on TLS. Moreover, we found that the colony forming potential of TLS-dependent cancer cells is dramatically reduced upon inhibition of TLS. Thus, we propose to identify the scope of TLS dependent cancers using cell screening and data base analysis. In addition, to fully further develop the therapeutic potential of TLS inhibition, we propose to measure and improve the anti- cancer potential of our lead small molecules. Collectively, these proposed studies will identify how cancer cells engage TLS and how best to block TLS to selectively target cancer.